Production of Nisin Z by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> Isolated from Dahi

Lactococcus lactis CM1, an isolate from homemade “Dahi,” a traditional fermented milk from India, used maltose as carbon source to produce a high level of bacteriocin. The bacterial cell mass and the bacteriocin production correlated with the initial pH of the medium and were highest when the initial pH was 11.0. The level of bacteriocin reached its peak at the late log phase with concomitant reduction of culture pH to 4.2, regardless of the initial pH of the medium. A combination of maltose and an initial medium pH of 11 resulted in the highest bacteriocin production. The antibacterial spectrum of the bacteriocin was closely similar to that of nisin and it inhibited a number of food spoilage and pathogenic bacteria. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the compound migrated close to the position of nisin (3.5 kDa). However, it had higher stability than nisin at a wide range of pH and temperature. PCR amplification using nisin gene-specific primers and sequencing of the amplified DNA revealed the structural gene for the bacteriocin to be identical to that of nisZ.     

Stress Response Kinetics of Two Nisin Producer Strains of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis><Emphasis Type="Italic">-</Emphasis>(+)-Lactic Acid Production by an Adapted Strain of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using Corncob Hydrolysate

Corncob is an economic feedstock and more than 20 million tons of corncobs are produced annually in China. Abundant xylose can be potentially converted from the large amount of hemicellulosic materials in corncobs, which makes the crop residue an attractive alternative substrate for a value-added production of a variety of bioproducts. Lactic acid can be used as a precursor for poly-lactic acid production. Although current industrial lactic acid is produced by lactic acid bacteria using enriched medium, production by Rhizopus oryzae is preferred due to its exclusive formation of the l-isomer and a simple nutrition requirement by the fungus. Production of l-(+)-lactic acid by R. oryzae using xylose has been reported; however, its yield and conversion rate are poor compared with that of using glucose. In this study, we report an adapted R. oryzae strain HZS6 that significantly improved efficiency of substrate utilization and enhanced production of l-(+)-lactic acid from corncob hydrolysate. It increased l-(+)-lactic acid final concentration, yield, and volumetric productivity more than twofold compared with its parental strain. The optimized growth and fermentation conditions for Strain HZS6 were defined.     

Functionalized Alginate as Immobilization Matrix in Enantioselective <Emphasis Type="SmallCaps">l</Emphasis> (+) Lactic Acid Production by <Emphasis Type="Italic">Lactobacillus delbrucekii</Emphasis>

The imperative role of functionalized natural alginate in immobilization of Lactobacillus delbrucekii (NCIM 2365) cells in production of optically pure L (+) lactic acid was studied. L. delbrucekii cells were immobilized in alginate, succinylated alginate and carrageenan to evaluate the bead stability and selectivity towards production of optically pure L (+) lactic acid. The scanning electron microscopic studies of free and immobilized cells show little morphological changes. The present study highlights the use of functionalized alginate-immobilized L. delbrucekii cells in production of L (+) lactic acid in higher yields (0.93 Yp/s in grams) with an improved enantioselectivity (99%). In addition, they further revealed decreased by-product formation (acetic and propionic acid) when compared to free and other immobilized cell fermentation.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

A new 3,4-dihydroisocoumarin isolated from <Emphasis Type="Italic">Botryosphaeria</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> F00741

A new 3,4-dihydroisocoumarin, named botryoisocoumarin A (1), was obtained from the fermentation culture of Botryosphaeria sp. F00741, together with two known ones, 3,8-dihydroxy-3-methylisochroman-1-one (2) and 4,8-dihydroxy-3-methylisochroman-1-one (3). Their structures were elucidated by spectroscopic analyses, including 1D and 2D NMR experiments and ESI mass spectrometry, and by comparison with those reported. Compound 1 was evaluated for its antitumor and antimicrobial activities in vitro.     

Enzyme Production by Solid Substrate Fermentation of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and <Emphasis Type="Italic">Trametes versicolor</Emphasis> on Tomato Pomace

A process of solid state fermentation (SSF) on tomato pomace was developed with the white-rot fungi Pleurotus ostreatus and Trametes versicolor, using sorghum stalks as support. Operative parameters (humidity, water activity, and size of substrate particles) guaranteeing a good colonization of tomato pomace by both fungi were defined and conditions for production at high titers of the industrially relevant enzymes laccase, xylanase and protease were identified. Significant laccase activity levels (up to 36 U g⁻¹ dry matter) were achieved without any optimization of culture conditions, neither by nutrient addition nor by O₂ enrichment. Furthermore, protease activity levels up to 34,000 U g⁻¹ dry matter were achieved, being higher than those reported for the fungi typically considered as the best protease producers such as Aspergillus strains. Moreover, as one of the most significant results of this study, analysis of P. ostreatus tomato SSF samples by zymogram revealed two bands with laccase activity which had not been detected so far.     

A new ten-membered lactone from <Emphasis Type="Italic">Tubercularia</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> TF5, an endophytic fungus of <Emphasis Type="Italic">Taxus mairei</Emphasis>

One new ten-membered lactone (1) named (Z)-4,6,9-trihydroxy-10-nonyl-3,4,5,6,9,10-hexahydrooxecin-2-one along with 5-methoxycarbonylmellein (2) and cytochalasin D (3) were isolated from the culture of the endophytic fungus strain Tubercularia sp. TF5, originally separated from the inner bark of Taxus mairei obtained in Fujian Province, Southeast China. The structures of compounds 1–3 were elucidated by spectroscopic methods. The antimicrobial and cytotoxic activities of 1 were analyzed but it showed no significant activities.     

Copolymer of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride with maleic acid as drug carrier

A copolymer of N,N-diallyl-N,N-dimethylammonium chloride with maleic acid of constant composition was prepared under the conditions of radical initiation. The possibility of the functionalization of the copolymer with drugs containing amino groups by polymer-analogous transformations was examined. Conditions were found for preparing conjugates of the copolymer with isoniazid. The structures and the quantitative compositions of the conjugates were determined by ¹³С NMR spectroscopy, and the possibility of preparing conjugates with controlled drug content was demonstrated.     

Phosphorylation of <Emphasis Type="Italic">N</Emphasis>-glycosides derived from <Emphasis Type="Italic">para</Emphasis>-substituted aromatic amines

New carbohydrate-containing aminophosphoryl compounds were synthesized on the basis of glycosylamines. The reaction pathway suggested for addition of hydrophosphoryl compounds to N-glycosides was confirmed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1986–1992, September, 2008.     

Production of Anti-Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus</Emphasis> Activity from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> sp. Strain B38 Newly Isolated from Soil

B38 bacterial strain, isolated from Tunisian soil showed a strong antimicrobial activity. Based on biochemical characterization and 16S rDNA sequence analysis, B38 strain was identified as Bacillus subtilis. Cell culture supernatant showed antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus species and several Gram-positive and Gram-negative bacteria. Antifungal activity against phytopathogenic fungi was also observed. Antibacterial activity production started at early exponential growth phase, and maximum activity was reached at the stationary phase. This antibacterial activity was neither affected by proteases, lipase, and organic solvents, nor by surfactants. It was stable over a wide pH range and still active after autoclaving at 121 °C during 20 min. Thin layer chromatography followed by bioautography assay allowed the detection of four active spots with R _f values of 0.30, 0.47, 0.70, and 0.82. The single spot with R _f 0.30 showed antifungal activity, whereas the spots with R _f values of 0.47, 0.70, and 0.82 exhibited antibacterial activity.     

Molecular properties of the copolymers of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride and maleic acid

The hydrodynamic and conformational properties of molecules of poly(N,N-diallyl-N,N-dimethylammonium chloride) and N,N-diallyl-N,N-dimethylammonium chloride-maleic acid copolymers of different compositions in solutions with various ionic-strength and pH values, as well as of the polyelectrolyte complex based on the copolymer with dodecyl sulfate anions in chloroform, are studied. For poly(N,N-diallyl-N,N-dimethylammonium chloride) molecules in a 1 M NaCl solution, the Kuhn segment length and the hydrodynamic diameter of the chain are estimated as A = 3.9 nm and d = 0.48 nm, respectively. In acidic solutions with pH 3.5, the copolymers demonstrate behavior typical for polyelectrolytes. In an alkaline solution with pH 13, when 1 M NaCl is added to the solution of the copolymer containing 29 mol % maleic acid units, there is an antipolyelectrolyte effect that manifests itself as an increase in the intrinsic viscosity of the copolymer and in the hydrodynamic radius of its molecules. It is found that an increase in the fraction of maleic acid units in the copolymer from 12 to 42 mol % brings about a reduction in the equilibrium rigidity of its macromolecules from 4.1 to 2.2 nm. The equilibrium rigidity of polyelectrolyte-complex molecules is higher than that of initial copolymer molecules owing to steric interactions arising between the aliphatic chains of dodecyl sulfate anions. In an electric field, the molecules of the complex are oriented owing to the induced dipole moment resulting from the displacement of dodecyl sulfate anions along the chain contour.     

Heterologous Expression of Aldehyde Dehydrogenase in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> for Acetaldehyde Detoxification at Low pH

Aldehyde dehydrogenase (E.C. 1.2.1.x) can catalyze detoxification of acetaldehydes. A novel acetaldehyde dehydrogenase (istALDH) from the non-Saccharomyces yeast Issatchenkia terricola strain XJ-2 has been previously characterized. In this work, Lactococcus lactis with the NIsin Controlled Expression (NICE) System was applied to express the aldehyde dehydrogenase gene (istALDH) in order to catalyze oxidation of acetaldehyde at low pH. A recombinant L. lactis NZ3900 was obtained and applied for the detoxification of acetaldehyde as whole-cell biocatalysts. The activity of IstALDH in L. lactis NZ3900 (pNZ8148-istALDH) reached 36.4 U mL^?1 when the recombinant cells were induced with 50 ng mL^?1 nisin at 20 °C for 2 h. The IstALDH activity of recombinant L. lactis cells showed higher stability at 37 °C and pH 4.0 compared with the crude enzyme. L. lactis NZ3900 (pNZ8148-istALDH) could convert acetaldehyde at pH 2.0 while the crude enzyme could not. Moreover, the resting cells of L. lactis NZ3900 (pNZ8148-istALDH) showed a 2.5-fold higher activity and better stability in catalyzing oxidation of acetaldehyde at pH 2.0 compared with that of Escherichia coli expressing the IstALDH. Taken together, the L. lactis cells expressing recombinant IstALDH are potential whole-cell biocatalysts that can be applied in the detoxification of aldehydes.     

Conformations and properties of <Emphasis Type="Italic">O</Emphasis>-Alkyl-<Emphasis Type="Italic">S</Emphasis>-(2-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dialkylamino)-ethylmethylthiophosphonates

Conformers of the biologically active compounds CH₃P(O)(OR)(SCH₂CH₂NR ₂ ^′ ), where (I) R = i-C₄H₉, R′ = C₂H₅ and (II) R = C₂H₅, R′ = i-C₃H₇, are calculated within the AM1 level of theory. The elongated and twisted forms with maximum and minimum distances between a nitrogen atom and those of a phosphorus tetrahedron, respectively, and bearing a syn and anti oriented alkoxy group relative to a phosphoryl oxygen, are studied. It is found that the differences between the energy, electronic, and geometric parameters of these forms are apparent in differences between their properties, e.g., the ability to participate in complexation and protonation, reactions that to some extent simulate the interaction between a substance and a biological object.     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.     

Research on the Solid State Fermentation of Jerusalem Artichoke Pomace for Producing <Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">R</Emphasis>-2,3-Butanediol by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> ZJ-9

R,R-2,3-butanediol (R,R-2,3-BD) was produced by Paenibacillus polymyxa ZJ-9, which was capable of utilizing inulin without previous hydrolysis. The Jerusalem artichoke pomace (JAP) derived from the conversion of Jerusalem artichoke powder into inulin extract, which was usually used for biorefinery by submerged fermentation (SMF), was utilized in solid state fermentation (SSF) to produce R,R-2,3-BD. In this study, the fermentation parameters of SSF were optimized and determined in flasks. A novel bioreactor was designed and assembled for the laboratory scale-up of SSF, with a maximum yield of R,R-2,3-BD (67.90 g/kg (JAP)). This result is a 36.3% improvement compared with the flasks. Based on the same bath of Jerusalem artichoke powder, the total output of R,R-2,3-BD increased by 38.8% for the SSF of JAP combined with the SMF of inulin extraction. Overall, the utilization of JAP for R,R-2,3-BD production was beneficial to the comprehensive utilization of Jerusalem artichoke tuber.     

Improvement of <Emphasis Type="Italic">Saccharopolyspora spinosa</Emphasis> and the Kinetic Analysis for Spinosad Production

In this paper, a new spinosad-producing mutant UV-42-13 was obtained by employing rhamnose and sodium propionate resistant selection strategies in series with UV irradiation. Spinosad production of the mutant was 125.3 mg/L, improved 285.5% compared with that of the wild strain (32.5 mg/L).The results of experiment on tolerance of propyl alcohol addition showed that the tolerant ability to precursor was higher. The precursor-resistant ability of the mutant improved through tolerance experiment by adding propyl alcohol, and the spinosad production was greatly increased. The kinetic models for biomass, substrate consumption, and spinosad production of mutant strain and wild strain were studied by conducting batch fermentation in the shaking flask. The result showed that the kinetic models could describe the fermentation process of spinosad producing well.     

An Improved Stereocontrolled Route to <Emphasis Type="Italic">cis</Emphasis>-Erythrinanes by Combined Intramolecular <Emphasis Type="Italic">Strecker</Emphasis> and <Emphasis Type="Italic">Bruylants</Emphasis> Reaction

Summary. Condensation of (2-iodophenyl)ethylamines with cyclohexanoylacetaldehyde provided the corresponding aldimines which were reduced yielding secondary phenethylcyclohexanoylethylamines. These in turn were appropriate intermediates to prepare several erythrinanes by a sequential intramolecular Strecker and intramolecular Bruylants reaction. In contrast, the C-ring homologue schelhammeranes were not available on this route.Part of PhD thesis, LMU München, D     

Synthesis of <Emphasis Type="Italic">N,N</Emphasis>-disubstituted (<Emphasis Type="Italic">Z</Emphasis>)-<Emphasis Type="Italic">O</Emphasis>-methylnicotinamide oximes

A number of (Z)-N,N-dialkyl- and (Z)-N-acyl-N-alkyl-O-methylnicotinamide oximes was synthesized. Their configuration was confirmed by the NOESY experiment. Evaluation of fungicidal activity of compounds obtained was performed.